Protein-protein interactions are an essential key in biological processes, from the replication and expression of genes to the morphogenesis of organisms. Protein-protein interactions govern, amongst other things, ligand-receptor interaction and the subsequent signaling pathway; they are important in assembly of enzyme subunits, in the formation of biological supramolecular structures such as ribosomes, filaments, and virus particles and in antigen-antibody interactions.
Researchers have developed several approaches in attempts to identify protein-protein interactions. Co-purification of proteins and co-immunoprecipitation were amongst the first techniques used. However, these methods are tedious and do not allow high throughput screening. A major breakthrough was obtained by the introduction of the genetic approaches, of which the yeast two-hybrid (Fields and Song, 1989) is the most important one. Several improvements and modifications of this system have been published. As an example, we can cite U.S. Pat. No. 5,637,463 that describes a method of detecting post-translational modification-dependent protein-protein interactions.
Approaches based on phage display do avoid the nuclear translocation. PCT International Publication No. WO 90/02809 describes how a binding protein can be displayed on the surface of a genetic package, such as a filamentous phage, whereby the gene encoding the binding protein is packaged inside the phage. Phages, which bear the binding protein that recognizes the target molecule, are isolated and amplified. Several improvements of the phage display approach have been proposed, as described, for example, in PCT International Publication Nos. WO 92/20791, WO 97/10330, and WO 97/32017.
Another technique for assessing protein-protein interactions is based on fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) (PCT International Publication No. WO 99/66324).
Although in principle, all of those methods can be used in a positive (detecting protein-protein interactions) or a negative (detection of disruption of the protein-protein interaction) way, due to the positive selection in case of protein-protein interaction, none of these methods is really suited for screening of compounds that inhibit protein-protein interaction. To overcome this problem, U.S. Pat. No. 5,733,726 discloses a cytotoxicity-based genetic selection method, whereby a classic two-hybrid system is linked to the transcriptional activation of a toxic reporter gene, providing an assay for positive selection of mutations or small molecules or drugs disruptive for protein-protein interaction. An adaptation of this method has been disclosed in PCT International Publication No. WO 98/13502, whereby the protein-protein interaction induces the transcription of a repressor gene, which represses a selectable marker. Upon disruption of the protein-protein interaction, the repressor will no longer be synthesized, resulting in an activation of the selectable marker.